CN114836369B

CN114836369B - Method for differentiating induced pluripotent stem cells into pancreatic islets and application of induced pluripotent stem cells in treatment of type I diabetes

Info

Publication number: CN114836369B
Application number: CN202210556334.1A
Authority: CN
Inventors: 安翠平; 顾雨春
Original assignee: Chengnuo Regenerative Medical Technology Beijing Co ltd
Current assignee: Chengnuo Regenerative Medical Technology Beijing Co ltd
Priority date: 2022-05-20
Filing date: 2022-05-20
Publication date: 2023-01-17
Anticipated expiration: 2042-05-20
Also published as: WO2023221565A1; CN114836369A

Abstract

The invention discloses a method for differentiating induced pluripotent stem cells into pancreatic islets and application thereof in treating type I diabetes.

Description

Method for differentiating induced pluripotent stem cells into pancreatic islets and application of induced pluripotent stem cells in treatment of type I diabetes

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a method for inducing differentiation of pluripotent stem cells into pancreatic islets and application of the induced differentiation to treatment of type I diabetes.

Background

Diabetes mellitus is a disease characterized by hyperglycemia, and is caused by deficiency of insulin secretion or insulin resistance, which in turn causes disorder of glucose metabolism, due to various pathogenic factors. Currently, diabetes is classified into type i diabetes, type ii diabetes, specific type diabetes, and gestational diabetes. Among them, type I diabetes patients account for about 10% of diabetes patients, type I diabetes is an organ-specific autoimmune disease mediated by T lymphocytes and characterized by selective destruction of islet β cells, type I diabetes patients are absolutely deficient in insulin secretion themselves and need to rely on insulin for treatment for life, and as the condition progresses, patients eventually enter the "fragile diabetes stage". In addition, patients with type I diabetes have relatively insufficient insulin secretion due to destruction of pancreatic beta cells, which in turn exposes multiple organs of the body to a hyperglycemic environment for a long time, resulting in severe cardiovascular, ocular, renal and nervous system pathologies. Serious complications can occur if the treatment is improper or untimely, the life quality of the patient is influenced by mild people, and the death of the patient is caused by severe people.

At present, the standard treatment scheme of the type I diabetes is drug therapy, namely, the modes of drug therapy such as oral hypoglycemic drugs, insulin injection, insulin pumps and the like are adopted, but the uninterrupted high-quality insulin injection therapy is required to be ensured for the whole life of a patient. Type I diabetes patients cannot be cured at present, and although the glucose metabolism can be effectively improved and the life quality of the patients can be improved by means of drug therapy, the risk of acute complications such as infection, ketoacidosis and hypoglycemia and chronic complications such as retina, nerve, kidney, cardiovascular and cerebrovascular diseases and large vascular plaques is increased by means of lifetime drug therapy. Therefore, long-term exogenous insulin injection can only relieve the hyperglycemia symptoms of diabetes, but cannot fundamentally treat the diabetes, thereby obtaining good intervention and treatment effects. Therefore, there is a need in the art for a new and effective therapeutic strategy that can reduce or even eliminate acute and chronic complications for the treatment of type i diabetes.

In recent years, with the continuous development of islet transplantation and stem cell transplantation technologies, a new method is provided for treating type I diabetes. Although islet transplantation is an ideal method for treating type I diabetes, the clinical application of the method is limited because islet transplantation cannot be performed in most patients due to the problems of lack of donor sources, immune rejection after transplantation and the like. In recent years, stem cell transplantation has been increasingly used for the treatment of diabetes. However, the problem with this approach is that the source of islet beta cells that secrete insulin, the only blood glucose-lowering hormone in the body, is insufficient and the blood glucose regulation function in the body is not maintained for a long time. Relevant research shows that the functional insulin secreting cell obtained through stem cell inducing differentiation, reprogramming and transdifferentiation technology has wide foreground. The embryonic stem cells have the characteristics of unlimited proliferation capacity, multidirectional differentiation potential and the like, however, the clinical application of the embryonic stem cells is limited by ethical reasons at present, so that Induced pluripotent stem cells (iPSCs) become seed cells which can provide sources for secreting insulin indefinitely, and potential medical ethical problems can be avoided.

The latest research shows that the induced pluripotent stem cells not only can generate specific cell types for patients, but also opens up a new way for innovations such as disease treatment, drug screening, regenerative medicine and the like. The induced pluripotent stem cell is used as a stem cell with the multidirectional differentiation potential, and relatively mature and functional tissues or organs can be obtained in vitro through the induction of related chemical small molecules and cell growth factors. The method for obtaining tissues and organs by utilizing the directional differentiation of stem cells can also be used for treating diabetes, and provides a new solution for donor deficiency. However, differentiation of pluripotent stem cells into pancreatic endocrine cells is currently achieved primarily through the combined use of signaling molecules and their associated inhibitors/agonists, typically following 6-7 sequential differentiation stages, respectively: definitive endoderm, primitive embryonic gut tube, posterior foregut, pancreatic endoderm, endocrine precursor cells and beta-like early cells, and further differentiate into mature islet beta-like cells. However, the differentiation method still produces immature beta cells, the single positive rate of GCG-/INS + cells is generally low, the expressed hormones are limited and unstable, the types are various, the content of insulin is limited, and the method cannot be used for transplanting and treating the diabetic patients.

It can be seen that, since insulin injection causes various complications, the source of islet transplantation is limited, and the currently developed islet beta cell differentiation protocol is immature, the number of islet beta cell is small, the single positive rate of GCG-/INS + cells is generally low, or the currently developed islet beta cell differentiation protocol does not have the function of glucose-stimulated insulin secretion (GSIS), so that the diabetic cell therapy is greatly limited, and therefore, how to obtain a large amount of functional, mature and stable islet beta cells from induced pluripotent stem cells is one of the main problems facing the field of type I diabetic cell therapy.

Disclosure of Invention

In order to solve the above problems faced by the present field, the present invention aims to provide a method for inducing differentiation of pluripotent stem cells into pancreatic islets and its use in the treatment of type I diabetes.

The above purpose of the invention is realized by the following technical scheme:

the first aspect of the invention provides an induction differentiation agent for inducing iPSCs to differentiate into functionally mature islet beta cells.

Further, the induced differentiation agent comprises a first stage induced differentiation agent, a second stage induced differentiation agent, a third stage induced differentiation agent, a fourth stage induced differentiation agent, a fifth stage induced differentiation agent, a sixth stage induced differentiation agent and a seventh stage induced differentiation agent;

preferably, the first-stage induction differentiation agent comprises a first-stage induction differentiation agent A and a first-stage induction differentiation agent B;

more preferably, the first stage induction differentiation agent a comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, activin a, CHIR-99021;

more preferably, the first stage induction differentiation agent B comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, activin a;

preferably, the second stage induction differentiation agent comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ascorbic acid, FGF-7;

preferably, the third stage induction differentiation agent comprises sodium bicarbonate, glutamine, glucose, bovine serum albumin, ascorbic acid, FGF-7, insulin-transferrin-selenium, ethanolamine, SANT-1, retinol, LDN193189, TPPB;

preferably, the fourth stage induction differentiation agent comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ascorbic acid, FGF-7, insulin-transferrin-selenium, ethanolamine, SANT-1, retinol, LDN193189, TPPB;

preferably, the fifth stage induction differentiation agent comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, SANT-1, retinol, LDN193189, triiodothyronine (T3), ALK5 iii, zinc sulfate;

preferably, the sixth stage induction differentiation agent comprises a sixth stage induction differentiation agent a, a sixth stage induction differentiation agent B, a sixth stage induction differentiation agent C and a sixth stage induction differentiation agent D;

more preferably, the sixth stage induction differentiation agent a comprises betacellulin, latrunculin a;

most preferably, the sixth stage induction differentiation agent a further comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, LDN193189, triiodothyronine (T3), ALK5i II, zinc sulfate, GSi XX;

more preferably, said sixth stage induction differentiation agent B comprises betacellulin;

most preferably, the sixth stage induction differentiation agent B further comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, LDN193189, triiodothyronine (T3), ALK5i II, zinc sulfate, GSi XX;

more preferably, said sixth stage induction differentiation agent C comprises betacellulin, forskolin, exenatide 4;

most preferably, the sixth stage induction differentiation agent C further comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, LDN193189, triiodothyronine (T3), ALK5i II, zinc sulfate, GSi XX;

more preferably, said sixth stage induction differentiation agent D comprises betacellulin, forskolin, exenatide 4, hepatocyte growth factor, 5 hydroxytryptamine;

most preferably, the sixth stage induction differentiation agent D further comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ethanolamine, LDN193189, triiodothyronine (T3), ALK5i II, zinc sulfate, GSi XX, insulin-transferrin-selenium;

preferably, the seventh stage induction differentiation agent comprises a seventh stage induction differentiation agent a, a seventh stage induction differentiation agent B;

more preferably, said seventh stage induction differentiation agent a comprises betacellulin, forskolin, exenatide 4, hepatocyte growth factor, 5 hydroxytryptamine;

most preferably, the seventh stage induction differentiation agent a further comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, triiodothyronine (T3), ALK5i II, zinc sulfate, N-acetyl-L-cysteine, porcine intestinal mucosal heparin sodium, water soluble vitamins E, R;

more preferably, said seventh stage induction differentiation agent B comprises glutamine, calcium chloride dihydrate, N-2 hydroxyethylpiperazine-N-2-ethanesulfonic acid, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, water-soluble vitamin E, niacinamide, heparin sodium, dnase i, necrostatin-1, pefabloc;

most preferably, the concentrations of the ingredients in the first-stage induced differentiation agent are respectively: (10-500) ng/mL Activin A, (0.01-10) μ M CHIR-99021, (0.01-10)% fetal bovine serum albumin, (0.01-10) g/L sodium bicarbonate, (1-50) mM glucose, (0.01-5) mM glutamine;

most preferably, the concentrations of the ingredients in the first-stage induced differentiation agent are respectively: 100 ng/mL Activin A, 3. Mu.M CHIR-99021, 0.5% bovine serum albumin, 1.5g/L sodium bicarbonate, 10 mM glucose, 1 mM glutamine;

more preferably, the concentrations of the ingredients in the second-stage induction differentiation agent are respectively: (0.01-10) mM ascorbic acid, (10-200) ng/mL FGF-7, (0.01-10)% bovine serum albumin, (0.01-10) g/L sodium bicarbonate, (1-50) mM glucose, (0.01-5) mM glutamine;

most preferably, the concentrations of the ingredients in the second stage induction differentiation agent are respectively: 0.25 mM ascorbic acid, 50 ng/mL FGF-7, 0.5% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, 10 mM glucose, 1 mM glutamine;

more preferably, the concentrations of the ingredients in the third stage induction differentiation agent are respectively: (0.01-5) mM ascorbic acid, (10-200) ng/mL FGF-7, (0.01-5) μ M SANT-1, (0.01-5) μ M retinol, (10-500) nM LDN193189, (50-500) nM TPPB, (0.01-10)% fetal bovine serum albumin, (0.01-10) g/L sodium bicarbonate, (1-100) mM glucose, (1-100) mM glutamine, (1-100) mg/L ethanolamine, (0.01-5)% insulin-transferrin-selenium;

most preferably, the concentrations of the ingredients in the third stage induction differentiation agent are respectively: 0.25 mM ascorbic acid, 50 ng/mL FGF-7, 0.25. Mu.M SANT-1, 1. Mu.M retinol, 100 nM LDN193189, 200 nM TPPB, 2% fetal bovine serum albumin, 2.5 g/L sodium bicarbonate, 10 mM glucose, 1 mM glutamine, 1 mg/L ethanolamine, 0.5% insulin-transferrin-selenium;

more preferably, the concentrations of the components in the fourth stage induction differentiation agent are respectively as follows: (0.01-5) mM ascorbic acid, (0.01-10) ng/mL FGF-7, (0.01-5) μ M SANT-1, (0.01-5) μ M retinol, (50-500) nM LDN193189, (10-300) nM TPPB, (0.01-5)% fetal bovine serum albumin, (0.05-10) g/L sodium bicarbonate, (0.05-50) mM glucose, (0.01-5) mM glutamine, (0.01-5) mg/L ethanolamine, (0.01-5)% insulin-transferrin-selenium;

most preferably, the concentrations of the components in the fourth stage induction differentiation agent are respectively as follows: 0.25 mM ascorbic acid, 2 ng/mL FGF-7, 0.25 μ M SANT-1, 0.1 μ M retinol, 200 nM LDN193189, 100 nM TPPB, 2% fetal bovine serum albumin, 2.5 g/L sodium bicarbonate, 10 mM glucose, 1 mM glutamine, 1 mg/L ethanolamine, 0.5% insulin-transferrin-selenium;

more preferably, the concentrations of the ingredients in the fifth stage induction differentiation agent are respectively as follows: (0.01-5) μ M SANT-1, (0.01-10) μ M retinol, (10-500) nM LDN193189, (0.01-5) μ M triiodothyronine (T3), (1-50) μ M ALK5 llI, (1-50) μ M zinc sulfate, (0.05-10)% fetal bovine serum albumin, (0.05-10) g/L sodium bicarbonate, (1-50) mM glucose, (0.01-10) mM glutamine, (0.01-5)% insulin-transferrin-selenium, and (0.01-10) mg/L ethanolamine;

most preferably, the concentrations of the ingredients in the fifth stage induction differentiation agent are respectively: 0.25 μ M SANT-1, 0.05 μ M retinol, 100 nM LDN193189, 1 μ M triiodothyronine (T3), 10 μ M ALK5i II,10 μ M zinc sulfate, 2% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, 20 mM glucose, 1 mM glutamine, 0.5% insulin-transferrin-selenium, 1 mg/L ethanolamine;

most preferably, the concentrations of the ingredients in the sixth stage induction differentiation agent are: (10-500) nM LDN193189, (0.01-10) μ M triiodothyronine (T3), (1-50) μ M ALK5i II, (1-50) μ M zinc sulfate, (0.05-10)% fetal bovine serum albumin, (0.01-5) g/L sodium bicarbonate, (1-100) mM glucose, (0.01-5) mM glutamine, (0.01-5) mg/L ethanolamine, (50-300) nM GSi XX, (0.01-5) μ M latrunculin A, (10-500) ng/mL hepatocyte growth factor, (1-50) μ M5 hydroxytryptamine, (1-50) ng/mL β -cell, (1-50) μ M forskolin, (10-500) ng/mL exenatide 4;

most preferably, the concentrations of the ingredients in the sixth stage induction differentiation agent are: 100 nM LDN193189, 1. Mu.M triiodothyronine (T3), 10. Mu.M ALK5i II, 10. Mu.M zinc sulfate, 2% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, 20 mM glucose, 1 mM glutamine, 1 mg/L ethanolamine, 100 nM GSi XX, 1. Mu.M latrunculin A, 50 ng/mL hepatocyte growth factor, 10. Mu.M 5 hydroxytryptamine, 20 ng/mL betacellulin, 10. Mu.M forskolin, 50 ng/mL exenatide 4;

most preferably, the concentrations of the ingredients in the seventh stage induction differentiation agent a are respectively: (0.05-10)% fetal bovine serum albumin, (0.05-10) g/L sodium bicarbonate, (5-50) mM glucose, (0.01-5) mM glutamine, (0.01-5) mg/L ethanolamine, (0.01-2.5)% insulin-transferrin-selenium, (0.01-5) μ M triiodothyronine (T3), (0.01-50) μ M ALK5i II, (1-50) μ M zinc sulfate, (0.01-5) mM N-acetyl-L-cysteine, (1-50) μ g/mL porcine intestinal mucosa heparin sodium, (1-50) μ M water-soluble vitamin E, (1-50) M R428 8, (10-100) ng/mL hepatocyte growth factor, (1-50) μ M5 hydroxytryptamine, (1-50) ng/mL beta-cellulin, (1-50) μ M laryngin, and (10-100) ng/mL exenatide 4;

most preferably, the concentrations of the ingredients in the seventh stage induction differentiation agent a are respectively: 2% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, 20 mM glucose, 1 mM glutamine, 1 mg/L ethanolamine, 0.5% insulin-transferrin-selenium, 1 μ M triiodothyronine (T3), 10 μ M ALK5 iii, 10 μ M zinc sulfate, 1 mM N-acetyl-L-cysteine, 10 μ g/mL porcine intestinal mucosal sodium heparin, 10 μ M water-soluble vitamin E, 2 μ M R, 50 ng/mL hepatocyte growth factor, 10 μ M5 hydroxytryptamine, 20 ng/mL beta cell, 10 μ M forskolin, 50 ng/mL exenatide 4;

most preferably, the concentrations of the ingredients in the seventh stage induction differentiation agent B are respectively as follows: (0.01-10) mM glutamine, (0.01-10) mM calcium chloride dihydrate, (1-50) mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, (0.01-5)% fetal bovine serum albumin, (0.01-2) mL/L insulin-transferrin-selenium, (0.01-5) mg/L ethanolamine, (1-50) μ M water-soluble vitamin E, (1-50) mM nicotinamide, (1-50) μ g/mL heparin sodium, (0.01-2.5) U/mL deoxyribonuclease I, (50-500) μ M Neocriptin-1, (0.001-2) μ M Pefabloc;

most preferably, the concentrations of the ingredients in the seventh stage induction differentiation agent B are respectively: 2 mM glutamine, 2.5 mM calcium chloride dihydrate, 10 mM N-2 hydroxyethyl piperazine-N-2 ethane sulfonic acid, 2% fetal bovine serum albumin, 0.6 mL/L insulin-transferrin-selenium, 1 mg/L ethanolamine, 10 μ M water soluble vitamin E, 10 mM nicotinamide, 10 μ g/mL heparin sodium, 1U/mL deoxyribonuclease I, 100 μ M Neostatin-1, 0.1 μ M Pefabloc.

In a second aspect, the invention provides an induced differentiation medium for inducing the differentiation of iPSCs into functionally mature islet beta cells.

Further, the induced differentiation culture medium comprises a first stage induced differentiation culture medium, a second stage induced differentiation culture medium, a third stage induced differentiation culture medium, a fourth stage induced differentiation culture medium, a fifth stage induced differentiation culture medium, a sixth stage induced differentiation culture medium and a seventh stage induced differentiation culture medium;

preferably, the first stage induced differentiation medium comprises a basal medium MCDB131, the first stage induced differentiation agent described in the first aspect of the present invention;

preferably, the second stage induced differentiation medium comprises the basal medium MCDB131, the second stage induced differentiation agent described in the first aspect of the present invention;

preferably, the third stage induced differentiation medium comprises the basal medium MCDB131, the third stage induced differentiation agent as described in the first aspect of the present invention;

preferably, the fourth stage differentiation-inducing medium comprises the basal medium MCDB131, the fourth stage differentiation-inducing agent as described in the first aspect of the present invention;

preferably, the fifth stage differentiation-inducing medium comprises the basal medium MCDB131, the fifth stage differentiation-inducing agent described in the first aspect of the present invention;

preferably, the sixth stage induced differentiation medium comprises a basal medium MCDB131, the sixth stage induced differentiation agent described in the first aspect of the present invention;

preferably, the seventh stage induced differentiation medium comprises the basal medium MCDB131, 50% Ham's F-12 medium, 50% medium 199, the seventh stage induced differentiation agent described in the first aspect of the present invention.

A third aspect of the invention provides a method of inducing iPSCs to differentiate into functionally mature pancreatic islet beta cells.

Further, the method comprises the steps of:

(1) Providing iPSCs and carrying out monolayer differentiation culture in a complete culture medium;

(2) Inducing differentiation at a first stage, namely inducing the iPSCs obtained in the step (1) to differentiate into definitive endoderm cells by adopting a first-stage induced differentiation culture medium;

(3) Inducing differentiation in the second stage, and inducing differentiation of definitive endoderm cells to primitive gut tube cells by adopting a second-stage inducing differentiation culture medium;

(4) Inducing differentiation in the third stage, namely inducing differentiation of the primitive gut tube cells to backward foregut cells by adopting a third-stage inducing differentiation culture medium;

(5) A fourth stage of induced differentiation, wherein the foregut cells are induced to differentiate into pancreatic progenitor cells by adopting a fourth stage of induced differentiation culture medium;

(6) Inducing differentiation in the fifth stage, namely inducing the pancreatic progenitor cells to differentiate into pancreatic endocrine progenitor cells by adopting a fifth-stage induced differentiation culture medium;

(7) Inducing differentiation in a sixth stage, namely inducing the pancreatic endocrine progenitor cells to differentiate into pancreatic endocrine cells by adopting a sixth-stage induced differentiation culture medium;

(8) And a seventh stage of induced differentiation, wherein a seventh stage of induced differentiation culture medium is adopted to induce pancreatic endocrine cells to differentiate into mature pancreatic islet cells, so as to obtain the functional mature pancreatic islet beta cells.

Further, the complete culture medium in the step (1) is an E8 complete culture medium;

preferably, the E8 complete medium contains a ROCK inhibitor;

more preferably, the ROCK inhibitor comprises Y27632, GSK429286A, RKI-1447, Y33075 dihydrochloride, thiazoviin, K-115, SLx-2119, chroman1, SAR407899, and/or SR-3677;

most preferably, the ROCK inhibitor is Y27632;

most preferably, the concentration of Y27632 is 0.001-100. Mu.M;

most preferably, the concentration of Y27632 is 10 μ M;

preferably, the iPSCs described in step (1) are derived from a mammal;

more preferably, the iPSCs described in step (1) are derived from human, mouse, rat, goat, sheep, pig, cat, rabbit, dog, wolf, horse or cow;

most preferably, the iPSCs described in step (1) are human in origin;

most preferably, the time of said culturing in step (1) is 3 days;

most preferably, day-3 to Day-1, with fresh medium changed daily;

preferably, the first stage differentiation induction medium described in step (2) comprises basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, activin, GSK-3 inhibitor;

more preferably, the Activin comprises Activin A, activin B, activin AB, activin AC;

most preferably, the Activin is Activin a;

more preferably, the GSK-3 inhibitor comprises CHIR-99021, CHIR-98014, AZD-2858, SB-216763, AT-7519, TW-S119, KY-19382 (A3051), NP-031112, SB-415286, AZD-1080, AR-A014418, TDZD-8, LY-2090314;

most preferably, the GSK-3 inhibitor is CHIR-99021;

more preferably, the first-stage differentiation-inducing medium includes a first-stage differentiation-inducing medium a and a first-stage differentiation-inducing medium B;

most preferably, the first stage differentiation induction medium A comprises the basic medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, activin A, CHIR-99021;

most preferably, the first stage differentiation induction medium B comprises the basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, activin a;

most preferably, the concentrations of the components in the first stage differentiation induction medium are respectively: (10-500) ng/mL Activin A, (0.01-10) μ M CHIR-99021, (0.01-10)% fetal bovine serum albumin, (0.01-10) g/L sodium bicarbonate, (1-50) mM glucose, (0.01-5) mM glutamine;

most preferably, the concentrations of the components in the first stage differentiation induction medium are: 100 ng/mL Activin A, 3. Mu.M CHIR-99021, 0.5% bovine serum albumin, 1.5g/L sodium bicarbonate, 10 mM glucose, 1 mM glutamine;

most preferably, the first stage induces differentiation for a period of 3 days;

most preferably, on the 1 st day of induction, the first stage induction differentiation medium A is used for induction differentiation;

most preferably, the first stage induction differentiation culture medium B is adopted for induction differentiation at the induction days 2-3;

most preferably, day0-Day2, fresh medium is replaced daily.

Further, the second stage differentiation induction medium in step (3) comprises a basic medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ascorbic acid and fibroblast growth factor;

preferably, the fibroblast growth factor comprises FGF-7, FGF-2, FGF-6, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-15, FGF-16, FGF-17, FGF-18, FGF-21, FGF-5, FGF-1, FGF-3, FGF-4, FGF-8, FGF-9, FGF-19, FGF-20;

more preferably, the fibroblast growth factor is FGF-7;

most preferably, the concentrations of the components in the second-stage differentiation-inducing medium are respectively: (0.01-10) mM ascorbic acid, (10-200) ng/mL FGF-7, (0.01-10)% bovine serum albumin, (0.01-10) g/L sodium bicarbonate, (1-50) mM glucose, (0.01-5) mM glutamine;

most preferably, the concentrations of the components in the second-stage differentiation-inducing medium are respectively: 0.25 mM ascorbic acid, 50 ng/mL FGF-7, 0.5% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, 10 mM glucose, 1 mM glutamine;

most preferably, the period of time for said second stage to induce differentiation is 2 days;

most preferably, on days 1-2 of induction, inducing differentiation is performed using the second-stage differentiation-inducing medium;

most preferably, day3-Day4, fresh medium is changed daily;

preferably, the third stage differentiation induction medium described in step (4) comprises a basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ascorbic acid, fibroblast growth factor, insulin-transferrin-selenium, ethanolamine, SANT-1, retinol, a BMP inhibitor, a protein kinase C activator;

more preferably, the fibroblast growth factor comprises FGF-7, FGF-2, FGF-6, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-15, FGF-16, FGF-17, FGF-18, FGF-21, FGF-5, FGF-1, FGF-3, FGF-4, FGF-8, FGF-9, FGF-19, FGF-20;

most preferably, the fibroblast growth factor is FGF-7;

more preferably, the BMP inhibitor comprises LDN193189, LDN212854, UK383367, LDN214117, GW788388, SM1-71, ER50891, DMH-1, LDN193189, K02288, PD161570;

most preferably, the BMP inhibitor is LDN193189;

more preferably, the protein kinase C activator comprises TPPB, PMA;

most preferably, the protein kinase C activator is TPPB;

most preferably, the concentrations of the components in the third stage differentiation-inducing medium are: (0.01-5) mM ascorbic acid, (10-200) ng/mL FGF-7, (0.01-5) μ M SANT-1, (0.01-5) μ M retinol, (10-500) nM LDN193189, (50-500) nM TPPB, (0.01-10) fetal bovine serum albumin, (0.01-10) g/L sodium bicarbonate, (1-100) mM glucose, (1-100) mM glutamine, (1-100) mg/L ethanolamine, (0.01-5)% insulin-transferrin-selenium;

most preferably, the concentrations of the components in the third stage differentiation-inducing medium are: 0.25 mM ascorbic acid, 50 ng/mL FGF-7, 0.25 μ M SANT-1, 1 μ M retinol, 100 nM LDN193189, 200 nM TPPB, 2% fetal bovine serum albumin, 2.5 g/L sodium bicarbonate, 10 mM glucose, 1 mM glutamine, 1 mg/L ethanolamine, 0.5% insulin-transferrin-selenium;

most preferably, the period of time for the third stage to induce differentiation is 2 days;

most preferably, on days 1-2 of induction, the third stage differentiation induction medium is used for induction differentiation;

most preferably, day5-Day6, fresh medium is replaced daily.

Further, the fourth stage differentiation induction medium described in the step (5) comprises a basal medium MCDB131, sodium bicarbonate, glutamine, glucose, bovine serum albumin, ascorbic acid, fibroblast growth factor, insulin-transferrin-selenium, ethanolamine, SANT-1, retinol, BMP inhibitor, protein kinase C activator;

more preferably, the fibroblast growth factor is FGF-7;

preferably, the BMP inhibitor comprises LDN193189, LDN212854, UK383367, LDN214117, GW788388, SM1-71, ER50891, DMH-1, LDN193189, K02288, PD161570;

more preferably, the BMP inhibitor is LDN193189;

preferably, the protein kinase C activator comprises TPPB, PMA;

more preferably, the protein kinase C activator is TPPB;

most preferably, the concentrations of the components in the fourth stage differentiation induction medium are respectively: (0.01-5) mM ascorbic acid, (0.01-10) ng/mL FGF-7, (0.01-5) μ M SANT-1, (0.01-5) μ M retinol, (50-500) nM LDN193189, (10-300) nM TPPB, (0.01-5)% fetal bovine serum albumin, (0.05-10) g/L sodium bicarbonate, (0.05-50) mM glucose, (0.01-5) mM glutamine, (0.01-5) mg/L ethanolamine, (0.01-5)% insulin-transferrin-selenium;

most preferably, the concentrations of the components in the fourth stage differentiation induction medium are respectively: 0.25 mM ascorbic acid, 2 ng/mL FGF-7, 0.25 μ M SANT-1, 0.1 μ M retinol, 200 nM LDN193189, 100 nM TPPB, 2% fetal bovine serum albumin, 2.5 g/L sodium bicarbonate, 10 mM glucose, 1 mM glutamine, 1 mg/L ethanolamine, 0.5% insulin-transferrin-selenium;

most preferably, the fourth stage induces differentiation for a period of 3 days;

most preferably, on days 1-3 of induction, the fourth stage induction differentiation medium is used for induction differentiation;

most preferably, day7-Day9, with fresh medium changed daily;

preferably, the fifth stage differentiation induction medium described in step (6) comprises basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, SANT-1, retinol, BMP inhibitor, thyroid hormone, ALK5 inhibitor, zinc sulfate;

most preferably, the BMP inhibitor is LDN193189;

more preferably, the thyroid hormones include triiodothyronine (T3), tetraiodothyronine (T4);

most preferably, the thyroid hormone is triiodothyronine (T3);

more preferably, the ALK5 inhibitor includes ALK5i II, R-268712, SB505124, GW788388, SD208, SB431542, ITD-1, LY2109761, A83-01, LY2157299, TGF-beta receptor inhibitor V, TGF-beta receptor inhibitor I, TGF-beta receptor inhibitor IV, TGF-beta receptor inhibitor VII, TGF-beta receptor inhibitor VIII, TGF-beta receptor inhibitor II, TGF-beta receptor inhibitor VI, and TGF-beta receptor inhibitor III;

most preferably, the ALK5 inhibitor is ALK5 iii;

most preferably, the concentrations of the components in the fifth stage differentiation induction medium are respectively: (0.01-5) μ M SANT-1, (0.01-10) μ M retinol, (10-500) nM LDN193189, (0.01-5) μ M triiodothyronine (T3), (1-50) μ M ALK5 iii, (1-50) μ M zinc sulfate, (0.05-10)% fetal bovine serum albumin, (0.05-10) g/L sodium bicarbonate, (1-50) mM glucose, (0.01-10) mM glutamine, (0.01-5)% insulin-transferrin-selenium, (0.01-10) mg/L ethanolamine;

most preferably, the concentrations of the components in the fifth stage differentiation induction medium are respectively: 0.25 μ M SANT-1, 0.05 μ M retinol, 100 nM LDN193189, 1 μ M triiodothyronine (T3), 10 μ M ALK5i II,10 μ M zinc sulfate, 2% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, 20 mM glucose, 1 mM glutamine, 0.5% insulin-transferrin-selenium, 1 mg/L ethanolamine;

most preferably, the fifth stage induces differentiation for a period of 3 days;

most preferably, on days 1-3 of induction, the fifth stage differentiation induction medium is used for induction differentiation;

most preferably, day10-Day12, fresh medium is replaced daily.

Further, the sixth stage differentiation induction medium described in step (7) comprises basal medium MCDB131, BMP inhibitor, thyroid hormone, ALK5 inhibitor, zinc sulfate, fetal bovine serum albumin, sodium bicarbonate, glucose, glutamine, ethanolamine, γ -secretase inhibitor, latrunculin a, hepatocyte growth factor, 5 hydroxytryptamine, β -cytokine, forskolin, exenatide 4;

more preferably, the BMP inhibitor is LDN193189;

preferably, the thyroid hormones include triiodothyronine (T3), tetraiodothyronine (T4);

more preferably, the thyroid hormone is triiodothyronine (T3);

preferably, the ALK5 inhibitor comprises ALK5i II, R-268712, SB505124, GW788388, SD208, SB431542, ITD-1, LY2109761, A83-01, LY2157299, TGF-beta receptor inhibitor V, TGF-beta receptor inhibitor I, TGF-beta receptor inhibitor IV, TGF-beta receptor inhibitor VII, TGF-beta receptor inhibitor VIII, TGF-beta receptor inhibitor II, TGF-beta receptor inhibitor VI, and TGF-beta receptor inhibitor III;

more preferably, the ALK5 inhibitor is ALK5 iii;

preferably, the gamma-secretase inhibitor comprises GSi XX, GSi IX, GSi XI, GSi XII, GSi XIII, GSi XIV, GSi XVI, GSi XIX, GSi XVII, GSi XXI, RO4929097, LY450139, MK-0752, BMS-708163, LY411575, LY3039478;

more preferably, the gamma-secretase inhibitor is GSi XX;

preferably, the sixth stage differentiation-inducing culture medium comprises a sixth stage differentiation-inducing culture medium a, a sixth stage differentiation-inducing culture medium B, a sixth stage differentiation-inducing culture medium C and a sixth stage differentiation-inducing culture medium D;

more preferably, said sixth stage differentiation induction medium a comprises basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, LDN193189, triiodothyronine (T3), ALK5i II, zinc sulfate, GSi XX, betacellulin, latrunculin a;

more preferably, said sixth stage differentiation-inducing medium B comprises basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, LDN193189, triiodothyronine (T3), ALK5i II, zinc sulfate, GSi XX, betacellulin;

more preferably, said sixth stage differentiation induction medium C comprises a basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, LDN193189, triiodothyronine (T3), ALK5i II, zinc sulfate, GSi XX, betacellulin, forskolin, exenatide 4;

more preferably, said sixth stage differentiation induction medium D comprises basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ethanolamine, LDN193189, triiodothyronine (T3), ALK5i II, zinc sulfate, GSi XX, insulin-transferrin-selenium, betacellulin, forskolin, exenatide 4, hepatocyte growth factor, 5 hydroxytryptamine;

most preferably, the concentrations of the components in the sixth stage differentiation induction medium are: (10-500) nM LDN193189, (0.01-10) μ M triiodothyronine (T3), (1-50) μ M ALK5i II, (1-50) μ M zinc sulfate, (0.05-10)% fetal bovine serum albumin, (0.01-5) g/L sodium bicarbonate, (1-100) mM glucose, (0.01-5) mM glutamine, (0.01-5) mg/L ethanolamine, (50-300) nM GSi XX, (0.01-5) μ M latrunculin A, (10-500) ng/mL hepatocyte growth factor, (1-50) μ M5 hydroxytryptamine, (1-50) ng/mL β -cell, (1-50) μ M forskolin, (10-500) ng/mL exenatide 4;

most preferably, the concentrations of the components in the sixth stage differentiation induction medium are respectively: 100 nM LDN193189, 1. Mu.M triiodothyronine (T3), 10. Mu.M ALK5i II, 10. Mu.M zinc sulfate, 2% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, 20 mM glucose, 1 mM glutamine, 1 mg/L ethanolamine, 100 nM GSi XX, 1. Mu.M latrunculin A, 50 ng/mL hepatocyte growth factor, 10. Mu.M 5 hydroxytryptamine, 20 ng/mL betacellulin, 10. Mu.M forskolin, 50 ng/mL exenatide 4;

most preferably, the sixth stage induces differentiation for a period of 14 days;

most preferably, on the 1 st day of induction, differentiation is induced using the sixth-stage differentiation induction medium a;

most preferably, on induction days 2-7, differentiation is induced using a sixth stage induction differentiation medium B;

most preferably, on induction days 8-9, differentiation is induced using a sixth stage induction differentiation medium C;

most preferably, on days 10-14 of induction, differentiation is induced using stage six induction differentiation medium D;

most preferably, day13-Day27, half a Day of fluid change;

preferably, the seventh stage differentiation-inducing medium described in step (8) comprises a seventh stage differentiation-inducing medium a and a seventh stage differentiation-inducing medium B;

more preferably, the seventh stage differentiation-inducing medium a comprises a basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, thyroid hormone, ALK5 inhibitor, zinc sulfate, N-acetyl-L-cysteine, heparin sodium of porcine intestinal mucosa, water-soluble vitamin E, AXL inhibitor, betacellulin, forskolin, exenatide 4, hepatocyte growth factor, 5 hydroxytryptamine;

most preferably, the thyroid hormones include triiodothyronine (T3), tetraiodothyronine (T4);

most preferably, the thyroid hormone is triiodothyronine (T3);

most preferably, the ALK5 inhibitors include ALK5i II, R-268712, SB505124, GW788388, SD208, SB431542, ITD-1, LY2109761, A83-01, LY2157299, TGF-beta receptor inhibitor V, TGF-beta receptor inhibitor I, TGF-beta receptor inhibitor IV, TGF-beta receptor inhibitor VII, TGF-beta receptor inhibitor VIII, TGF-beta receptor inhibitor II, TGF-beta receptor inhibitor VI, and TGF-beta receptor inhibitor III;

most preferably, the ALK5 inhibitor is ALK5 iii;

most preferably, the AXL inhibitors include R428, BMS-907351, BMS-777607, XL184, TP-0903, XL092, LDC1267, LY2801653, CEP-40783, RU-301, S49076, ONO-7475, ningetinib;

most preferably, the AXL inhibitor is R428;

most preferably, the concentrations of the ingredients in the seventh stage differentiation induction medium a are: (0.05-10)% fetal bovine serum albumin, (0.05-10) g/L sodium bicarbonate, (5-50) mM glucose, (0.01-5) mM glutamine, (0.01-5) mg/L ethanolamine, (0.01-2.5)% insulin-transferrin-selenium, (0.01-5) μ M triiodothyronine (T3), (0.01-50) μ M ALK5i II, (1-50) μ M zinc sulfate, (0.01-5) mM N-acetyl-L-cysteine, (1-50) μ g/mL porcine intestinal mucosa heparin sodium, (1-50) μ M water-soluble vitamin E, (1-50) M R428 8, (10-100) ng/mL hepatocyte growth factor, (1-50) μ M5 hydroxytryptamine, (1-50) ng/mL beta-cellulin, (1-50) μ M laryngin, and (10-100) ng/mL exenatide 4;

most preferably, the concentration of each component in the seventh stage differentiation-inducing medium a is: 2% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, 20 mM glucose, 1 mM glutamine, 1 mg/L ethanolamine, 0.5% insulin-transferrin-selenium, 1 μ M triiodothyronine (T3), 10 μ M ALK5 iii, 10 μ M zinc sulfate, 1 mM N-acetyl-L-cysteine, 10 μ g/mL porcine intestinal mucosal sodium heparin, 10 μ M water-soluble vitamin E, 2 μ M R, 50 ng/mL hepatocyte growth factor, 10 μ M5 hydroxytryptamine, 20 ng/mL beta cell, 10 μ M forskolin, 50 ng/mL exenatide 4;

more preferably, said seventh stage differentiation-inducing medium B comprises Ham's F-12 medium, medium 199, glutamine, calcium chloride dihydrate, N-2 hydroxyethylpiperazine-N-2-ethanesulfonic acid, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, water-soluble vitamin E, nicotinamide, sodium heparin, dnase i, necrotic apoptosis inhibitor, serine protease inhibitor;

most preferably, the necrotic apoptosis inhibitor comprises Necrostatin-1, necrostatin-2;

most preferably, the necrotic apoptosis inhibitor is Necrostatin-1;

most preferably, the serpin comprises Pefabloc, benzamidine, MBTI, PMSF, LBTI;

most preferably, the serpin is Pefabloc;

most preferably, the concentrations of the ingredients in the seventh stage differentiation-inducing medium B are: 50% Ham's F-12 medium, 50% medium 199, (0.01-10) mM glutamine, (0.01-10) mM calcium chloride dihydrate, (1-50) mM N-2 hydroxyethylpiperazine-N-2-ethanesulfonic acid, (0.01-5)% fetal bovine serum albumin, (0.01-2) mL/L insulin-transferrin-selenium, (0.01-5) mg/L ethanolamine, (1-50) μ M water-soluble vitamin E, (1-50) mM nicotinamide, (1-50) μ g/mL heparin sodium, (0.01-2.5) U/mL deoxyribonuclease I, (50-500) μ M Neocriptine-1, (0.001-2) μ M Pefabloc;

most preferably, the concentrations of the ingredients in the seventh stage differentiation-inducing medium B are: 50% Ham's F-12 medium, 50% medium 199, 2 mM glutamine, 2.5 mM calcium chloride dihydrate, 10 mM N-2 hydroxyethylpiperazine-N-2-ethanesulfonic acid, 2% fetal bovine serum albumin, 0.6 mL/L insulin-transferrin-selenium, 1 mg/L ethanolamine, 10. Mu.M water-soluble vitamin E, 10 mM nicotinamide, 10. Mu.g/mL heparin sodium, 1U/mL deoxyribonuclease I, 100. Mu.M Neocriptine-1, 0.1. Mu.M Pefabloc;

most preferably, the seventh stage induces differentiation for a period of 14 days;

most preferably, on induction days 1-8, the seventh stage induction differentiation medium A is used for induction differentiation;

most preferably, on days 9-11 of induction, said seventh stage differentiation-inducing medium B is used for inducing differentiation;

most preferably, day28-Day40, half a Day.

In a fourth aspect of the invention, there is provided an iPSCs-derived functional mature pancreatic islet beta cell or cell population.

Further, the cell or cell population is obtained by inducing differentiation using the method of the third aspect of the present invention;

preferably, the islet beta cell or cell population is a functional, stable, mature islet beta cell or cell population;

preferably, the single positive rate of the islet beta cells or cell population GCG-/INS + cells is 95%.

In a fifth aspect of the present invention, there is provided a pharmaceutical composition for the treatment and/or prevention of diabetes.

Further, the pharmaceutical composition comprises a cell or population of cells according to the fourth aspect of the invention;

preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or adjuvant;

preferably, the diabetes mellitus comprises type I diabetes, type II diabetes, specific type diabetes, gestational diabetes;

more preferably, the diabetes is type i diabetes.

A sixth aspect of the invention provides the use of any one of the following:

(1) The application of the combination of beta-cytomin, forskolin, exenatide 4, hepatocyte growth factor and 5 hydroxytryptamine in inducing iPSCs to differentiate into functional islet beta cells;

(2) The application of the induction differentiation agent in the first aspect of the invention in preparing an induction differentiation culture medium for inducing iPSCs to differentiate into functional islet beta cells;

(3) The application of the induction differentiation agent in inducing iPSCs to differentiate into functional islet beta cells is disclosed in the first aspect of the invention;

(4) The induced differentiation culture medium of the second aspect of the invention is applied to inducing iPSCs to differentiate into functional islet beta cells;

(5) Use of a cell or population of cells according to the fourth aspect of the invention in the manufacture of a medicament for the treatment and/or prophylaxis of diabetes;

(6) The use of a pharmaceutical composition according to the fifth aspect of the invention for the treatment and/or prevention of diabetes;

more preferably, the diabetes is type i diabetes.

Compared with the prior art, the invention has the advantages and beneficial effects that:

the invention provides a method for differentiating induced pluripotent stem cells into pancreatic islets and application thereof in treating type I diabetes, wherein the method uses a small molecule of latrunculin A for inhibiting a skeleton protein on day13, uses beta-cytomin, forskolin and exenatide 4 on day 20, uses hepatocyte growth factor HGF and 5 hydroxytryptamine on day 22, and uses 50% Ham's F-12 medium, 50% medium 199, glutamine, calcium chloride dihydrate, N-2 hydroxyethyl piperazine-N-2-ethanesulfonic acid, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, water-soluble vitamin E, nicotinamide, heparin, deoxyribonuclease I, necrotizing apoptosis inhibitor Necrostatin-1 and serine protease inhibitor Pefabloc on day 35.

Drawings

FIG. 1 is a graph showing the results of cell plating area screening and Matrigel gel concentration screening, in which A is a graph: graph of results obtained by plating 12-well plate, 6-well plate, and 10 cm dishes, respectively, panel B: graph of results of Matrigel gel concentration screening, panel C: a statistical graph of results of Matrigel gel concentration screening;

fig. 2 is a graph of the detection result of the first stage definitive endoderm induction, in which, a graph: statistical results of qPCR detection of endoderm markers SOX17, FOXA2 and CRCX4 mRNA expression levels of negative control group (iPSC, iPS), activin A group (Act-A) and GDF-8 group (GDF-8), FIG. B: results of immunofluorescence detection of endoderm markers FOXA2 positive rate of Activin group A (Act-A) and GDF-8 group (GDF-8);

FIG. 3 is a graph showing the results of the second-stage gastral stage induction test, in which A is: results of immunofluorescence detection of the positive rate of the primitive gut markers HNF1 beta and FOXA2, panel B: a result statistical chart of positive rates of the original intestinal canal markers HNF1 beta and FOXA2 detected by qPCR;

FIG. 4 is a graph showing the results of the detection of the induction of the hindgut phase in the third phase, wherein A is a graph: results of positive rates of PDX-1 and SOX9 markers in the hindgut stage by immunofluorescence assay, panel B: a result statistical chart of positive rates of markers PDX-1 and SOX9 in the post-foregut stage by qPCR detection;

FIG. 5 is a graph of the results of a fourth stage pancreatic progenitor stage induction assay, wherein panel A: results of positive rates of PDX-1 and SOX9 markers in the hindgut stage by immunofluorescence assay, panel B: a result statistical chart of positive rates of markers PDX-1 and SOX9 in the post-foregut stage by qPCR detection;

FIG. 6 is a graph showing the results of detection of stage-fifth-stage pancreatic endocrine progenitor cell induction in which the expression levels of pancreatic endocrine progenitor cell markers NKX6.1, NGN3, PDX-1 mRNA were detected by qPCR in the control group (iPSC, iPS) and the stage-fifth induction group (Islet);

FIG. 7 is a graph showing the results of examination of the stage induction of the sixth stage pancreatic endocrine cell and the stage induction of the seventh stage mature islet cell, A: results of qPCR detection of the expression levels of insulin cell markers INS, GCG and NKX6.1 mRNA in group A, group B and control group (iPSC, iPS), and B is shown in the following chart: and (3) a result graph of positive rates of islet cell markers INS, GCG, NKX6.1 and MAFA in the group A and the group B through immunofluorescence detection.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. As will be understood by those of ordinary skill in the art: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, biomaterials, etc. used in the following examples are commercially available unless otherwise specified.

Example 1 method for inducing differentiation of iPSCs into pancreatic islets

1. Test reagent and test material

The experimental reagents and experimental materials involved in the examples of the present invention are shown in tables 1 and 2, respectively.

2. iPSCs resuscitation

The Induced Pluripotent Stem Cells (iPSCs) are from Jianuo medical science and technology Limited company in Beijing and are prepared by the iPSCs preparation method described in patent 201910110768.7 previously applied by the company.

(1) According to the amount of the culture medium required for recovery, an E8 complete culture medium containing ROCki is prepared, 1 mu L of ROCki stock solution is added to each ml of the culture medium, and the culture medium is preheated.

(2) 10-15 mL of E8 complete medium was prepared and preheated to 37 ℃.

(3) Taking out 1 freezing tube from the liquid nitrogen tank, immediately putting into a hot water barrel with the temperature of 38-39 ℃, and continuously shaking back and forth for 60-90 s to completely melt the frozen cell suspension.

(4) Once the cells in the cryopreservation tube are completely thawed (liquid state), the cells are immediately taken out of the hot water bucket, the surface of the cryopreservation tube is thoroughly disinfected by 75% alcohol, and the frozen cryopreservation tube is placed in an ultra-clean workbench. When the frozen tube is about to melt, the centrifuge tube containing E8 culture medium preheated to 37 ℃ is sterilized by alcohol and then is placed in an ultra-clean workbench.

(5) Taking out the cell suspension in the freezing tube under strict aseptic operation condition, injecting the cell suspension into a preheated E8 culture medium 15 mL centrifugal tube, gently blowing and beating for 2-3 times, centrifuging for 200 g multiplied by 5 min, and discarding the supernatant after the centrifugation is finished.

(6) Adding culture medium containing ROCki, gently blowing and beating for 2-3 times, and taking the Matrigel coated well plate/culture flask out of the incubator. The coating solution was aspirated away and the cell suspension was added gently along the uncoated side (not directly onto the coating). Placing the culture dish in an incubator for culture.

Note that: during recovery, a container to be inoculated is selected according to the number of cells, generally 50-80 ten thousand cells are inoculated in a 6-well plate, and 80-100 ten thousand cells are inoculated in a T25 bottle. 100-200 ten thousand cells were seeded in T75 flasks.

3. Passages of iPSCs

(1) Preparing 0.5 mM EDTA working solution: 5 mL of DPBS is sucked into a new 15 mL centrifuge tube, 5 mu L of 0.5M EDTA stock solution is added, and the EDTA working solution is obtained after uniform mixing. The digestive juice is prepared as it is used.

(2) According to the culture medium amount required for passage, an E8 complete culture medium containing ROCki is prepared, and 1 mu L of ROCki stock solution is added to each milliliter of the culture medium.

(3) The well plate/flask to be passaged was removed from the incubator, the supernatant was aspirated and washed twice with DPBS (the amount of DPBS used per time was not less than the amount of original medium) for 1 min each time (DBPS was placed in the well/flask for 30-45 sec and then aspirated off during the washing).

(4) After adding EDTA working solution, about 6 mL EDTA working solution was added to the T75 flask, and the flask was incubated in an incubator for 5 min, during which time the cells were observed under a mirror, and when all the cells in the colony started to round and no larger clumps fell, the EDTA was gently aspirated without disturbing the cells.

(5) Adding an E8 complete culture medium to terminate digestion, and preferably, quickly dripping the culture medium when the digestion is terminated to ensure that the iPSCs fall off in the form of lumps. Repeated blowing of the cells is avoided, so that the cells are not blown into single cells and are kept into cell masses as much as possible.

(6) Balancing, centrifuging for 200 g,5 min, removing supernatant after centrifuging, gently shaking the bottom of the centrifuge tube, adding 5 mL E8 complete culture medium containing ROCKi for resuspension, gently blowing cell pellet with Pasteur dropper (blowing is not more than 3 times), inoculating cell suspension into T25 bottle coated with Matrigel, placing the culture bottle at 37 deg.C, and 5% CO ₂ And (5) standing and culturing in an incubator.

(7) After 24 h culture, the whole medium was changed with fresh E8 complete medium.

(8) After that, the whole liquid changing operation is carried out every day, and the passage is carried out when the cell confluence is about 70% -80%.

(9) The inoculation number of iPSCs cells is about 8000 cells/cm ² According to different iPSCs lines, the passage density can be adjusted, and the iPSCs cell interval passage time is 5-7 days.

4. Preparation of 60X Matrigel-coated cell culture plate

(1) The 12-well plate and 25 mL of DMEM \ F12 medium were placed in advance at-20 ℃ and pre-cooled for about 20 min.

(2) In an ice box, a 1.5 mL EP tube containing Matrigel was opened with the aid of a hemostat. Sterile operation is carried out, and hands are not required to touch the bottle, so that rewarming is prevented.

(3) Sucking 400 mu L of pre-cooled coating liquid into an EP tube filled with Matrigel, repeatedly blowing and beating, and sucking the supernatant back into a pre-cooled centrifuge tube. The process needs to be repeated for many times, so that the Matrigel can be dissolved quickly as much as possible without rewarming.

(4) Taking out the pre-cooled coating to be coated and placing the coating on an ice box. Uniformly mixing the coating liquid by using an electric pipettor, and coating by using a proper measuring device according to the following volume: 12 orifice plate: 1 mL per well.

(5) The coated culture flask/plate was placed at 37 ℃ in 5% CO ₂ Incubate overnight in the incubator.

5. Cell plating area screening

As shown by fig. 1, the higher the positive cell rate of the cell process fraction, the different culture areas were screened, including: the results of the respective plating of 12-well plate, 6-well plate and 10 cm showed that the larger the culture area, the less likely the cells to clump together and the best clumping effect of the 12-well plate (see fig. 1A).

6. Matrigel gel concentration screening

According to the method, the final observed islet yield is induced by adopting the 12-pore plate and the Matrigel with the concentration of 0.05-0.1 mg/mL (group A) and the 12-pore plate and the Matrigel with the concentration of 0.1-0.2 mg/mL (group B), and the groups A and B with different glue concentrations are adopted in the initial plate laying process, so that the induction yield is the highest when the Matrigel concentration is 0.1-0.2 mg/mL (see fig. 1B and fig. 1C), so far, the method forms an optimal initial induction mode, namely the 12-pore plate is used for inducing and adding 0.1-0.2 mg/mL Matrigel.

7. iPSC single layer differentiation establishment (Day-3 to Day-1)

This stage consisted of 2 mL of E8 complete medium, 10 μ M Y27632. From 37 ℃ with 5% CO ₂ Taking out a 12-pore plate paved with Matrigel-coated in a cell culture box, removing liquid, and adding 2 mL of E8 Medium +10 mu M Y27632 into each pore;

the amplification time is 3 days, and the concentration of Y27632 in the culture medium is 10 mu M on the amplification day 1; on induction days 2-3, the medium was changed to E8 complete medium only; fresh medium was changed daily.

8. The first stage is as follows: induction of definitive endoderm (Day 0-2)

The first stage is the differentiation of iPSC to definitive endoderm, and the first stage induced differentiation culture medium consists of basal medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin), activin A and CHIR-99021.

In this medium, the concentration of Activin A was 100 ng/mL, the concentration of CHIR-99021 was 3. Mu.M, the concentration of fetal bovine serum albumin was 0.5%, the concentration of sodium bicarbonate was 1.5g/L, the final concentration of glucose was 10 mM, and the concentration of glutamine was 1 mM.

The induction time of the first stage is 3 days, and on the induction 1 day, the concentration of Activin A in the culture medium is 100 ng/mL; concentration of CHIR-99021 is 3. Mu.M; on induction days 2-3, the concentration of Activin A in the medium was 100 ng/mL; namely: induction day1, a first stage induction differentiation medium a consisting of basal medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin), activin a, and CHIR-99021 was used; day 2-3 of induction, differentiation medium B was induced using a first stage consisting of basal medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin) and Activin a. Fresh medium was changed daily.

In this stage, the GDF-8 group was defined as group B, activin A (Act-A) was defined as group A, and the positive rate of endoderm cell markers OX17, FOXA2 obtained after induction was examined by qPCR.

9. And a second stage: induction of the gastral phase (Day 3-4)

The second stage is differentiation of definitive endoderm cells to primitive gut tube cells, and the second stage induction differentiation culture medium consists of a basic culture medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin), ascorbic acid and FGF-7.

In this medium, the ascorbic acid concentration was 0.25 mM, FGF-7 concentration was 50 ng/mL, fetal bovine serum albumin concentration was 0.5%, sodium bicarbonate concentration was 1.5g/L, glucose final concentration was 10 mM, and glutamine concentration was 1 mM.

The time for the second phase of induction was 2 days, and the culture medium was replaced with fresh medium every day on days 1-2 of induction.

10. And a third stage: induction of the posterior foregut phase (Day 5-6)

The third stage is differentiation of primitive gut tube cells to posterior foregut cells, and the third stage induction differentiation culture medium consists of a basic culture medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin), ascorbic acid, FGF-7, insulin-transferrin-selenium, ethanolamine, SANT-1, retinol, LDN193189 and TPPB.

In this medium, the concentration of ascorbic acid was 0.25 mM, FGF-7 was 50 ng/mL, SANT-1 was 0.25. Mu.M, retinol was 1. Mu.M, LDN193189 was 100 nM, TPPB was 200 nM, fetal bovine serum albumin was 2%, sodium bicarbonate was 2.5 g/L, glucose was 10 mM, glutamine was 1 mM, ethanolamine was 1 mg/L, and insulin-transferrin-selenium was 0.5%.

The time for the third stage induction was 2 days, with fresh medium being changed daily on induction days 1-2.

11. A fourth stage: induction of pancreatic progenitor stage (Day 7-9)

The fourth stage is differentiation of posterior foregut cells to pancreatic progenitor cells, and the fourth stage differentiation induction culture medium consists of a basic culture medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin), ascorbic acid, FGF-7, insulin-transferrin-selenium, ethanolamine, SANT-1, retinol, LDN193189 and TPPB.

In this medium, ascorbic acid was 0.25 mM, FGF-7 was 2 ng/mL, SANT-1 was 0.25. Mu.M, retinol was 0.1. Mu.M, LDN193189 was 200 nM, TPPB was 100 nM, fetal bovine serum albumin was 2%, sodium bicarbonate was 2.5 g/L, glucose was 10 mM, glutamine was 1 mM, ethanolamine was 1 mg/L, and insulin-transferrin-selenium was 0.5%.

The fourth stage of induction time was 3 days, in the induction 1-3 days, the fresh culture medium was changed daily.

12. The fifth stage: induction of pancreatic endocrine progenitor stage (Day 10-12)

The fifth stage is differentiation from pancreatic progenitor cells to pancreatic endocrine progenitor cells, and the fifth stage differentiation induction culture medium consists of a basic culture medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin), insulin-transferrin-selenium, ethanolamine, SANT-1, retinol, LDN193189, thyroid hormone (T3), ALK5i II and zinc sulfate.

In the culture medium, the concentration of SANT-1 is 0.25 μ M, the concentration of retinol is 0.05 μ M, the concentration of LDN193189 is 100 nM, the concentration of thyroid hormone (T3) is 1 μ M, the concentration of ALK5i II is 10 μ M, the concentration of zinc sulfate is 10 μ M, the concentration of fetal bovine serum albumin is 2%, the concentration of sodium bicarbonate is 1.5g/L, the final concentration of glucose is 20 mM, the concentration of glutamine is 1 mM, the concentration of insulin-transferrin-selenium is 0.5%, and the concentration of ethanolamine is 1 mg/L.

The induction time in the fifth stage was 3 days, and the fresh medium was changed daily on days 1 to 3 of induction.

13. The sixth stage: induction of pancreatic endocrine cell stage (Day 13-27)

The sixth stage is the differentiation of pancreatic endocrine progenitor cells into pancreatic endocrine cells, the induction time in this stage is 14 days, and the preparation work on the induction day1 is as follows:

(1) A24-well plate 400. Mu.M microplate was loaded with 500. Mu.L of the anti-hypopdsorbing liquid. Balancing 1300 g and centrifuging for 5 min; removing bubbles, and centrifuging again if bubbles exist in the micropores, and discarding the liquid;

(2) Rinsing each well with basal medium or DPBS, 24-well plate 2 mL, discarding the solution when used;

(3) Preparing TD digestive juice Tryple: DPBS (TD) =1:1;

(4) Washing the cells once by using DPBS, digesting for 5 min by using TD digestive juice, digesting 1 hole by using 500 mu L of 1 hole digestive juice, terminating by using M3 main culture, counting, estimating the total amount of the cells, and obtaining a 24-hole plate with about 1500-2000 cells in each micropore of a 400 mu M micropore plate;

(5) After all cells in a 12-hole plate are digested, quickly using a liquid suction pump to suck and discard digestive juice, using a gun head to suck a main culture medium, stopping one hole, collecting the digested cell in a 50 mL centrifuge tube, centrifuging for 5 min, then using full culture medium to suspend and spread the digested cell in an anti-adsorption coated micropore plate, placing the digested cell in an incubator to settle for 20 min,300 g and 5 min, placing the digested cell in the incubator after the centrifugation is finished, and changing the cell for half a day for 1 to 14 days, wherein each hole is 1 mL.

The sixth stage induced differentiation culture medium of the group A from 1 to 14 days in the present stage consists of a basic culture medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (bovine serum albumin), insulin-transferrin-selenium, ethanolamine, LDN193189, thyroid hormone (T3), ALK5i II, zinc sulfate and a gamma-secretase inhibitor;

the sixth stage induced differentiation culture medium of day1 in the group B consists of a basic culture medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin), insulin-transferrin-selenium, ethanolamine, LDN193189, thyroid hormone (T3), ALK5i II, zinc sulfate, gamma-secretagogue enzyme inhibitor, beta-cell hormone and latrunculin A;

the group B of the sixth stage induced differentiation medium of days 2-7 does not contain latrunculin A as same as day 1;

group B, day 8-9, sixth stage induced differentiation medium consisting of basal medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin), insulin-transferrin-selenium, ethanolamine, LDN193189, thyroid hormone (T3), ALK5i II, zinc sulfate, gamma secretase inhibitor, beta-cell, forskolin, exenatide 4;

group B, day 10-14, sixth stage differentiation induction medium consisted of basal medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin), ethanolamine, LDN193189, thyroid hormone (T3), ALK5 iii, zinc sulfate, gamma secretase inhibitor, insulin-transferrin-selenium, betacellulin, forskolin, exenatide 4, hepatocyte growth factor, 5 hydroxytryptamine.

In the culture medium, the concentration of LDN193189 is 100 nM, the concentration of thyroid hormone (T3) is 1 muM, the concentration of ALK5i II is 10 muM, the concentration of zinc sulfate is 10 muM, the concentration of fetal bovine serum albumin is 2%, the concentration of sodium bicarbonate is 1.5g/L, the final concentration of glucose is 20 mM, the concentration of glutamine is 1 mM, the concentration of ethanolamine is 1 mg/L, the concentration of gamma-secretase inhibitor is 100 nM, the concentration of latrunculin A is 1 muM, the concentration of hepatocyte growth factor is 50 ng/mL, the concentration of 5 hydroxytryptamine is 10 muM, the concentration of beta-cell factor is 20 ng/mL, the concentration of forskolin is 10 muM, and the concentration of exenatide 534 is 50 zxft 5364 mL.

14. And a seventh stage: induction of mature islet cell stage (Day 28-40)

The seventh stage is the differentiation of pancreatic endocrine cells into mature islet cells

The induction differentiation culture medium of the seventh stage of 1-11 days in the group A consists of a basic culture medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin), insulin-transferrin-selenium, ethanolamine, thyroid hormone, ALK5i II, zinc sulfate, N-acetyl-L-cysteine, porcine intestinal mucosa heparin sodium, and a water-soluble vitamin E, AXL inhibitor R428, and the culture medium is changed by liquid half a day.

The seventh stage induced differentiation culture medium of the 1 st to 8 th days in the group B of the stage consists of a basic culture medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin), insulin-transferrin-selenium, ethanolamine, thyroid hormone, ALK5i II, zinc sulfate, N-acetyl-L-cysteine, porcine intestinal mucosa heparin sodium, water-soluble vitamin E, AXL inhibitor R428, beta-cell hormone, forskolin, exenatide 4, hepatocyte growth factor and 5 hydroxytryptamine, and the liquid is changed half a day.

In the culture medium, the concentration of fetal bovine serum albumin is 2%, the concentration of sodium bicarbonate is 1.5g/L, the final concentration of glucose is 20 mM, the concentration of glutamine is 1 mM, the concentration of ethanolamine is 1 mg/L, the concentration of insulin-transferrin-selenium is 0.5%, the concentration of thyroid hormone is 1 μ M, the concentration of ALK5i II is 10 μ M, the concentration of zinc sulfate is 10 μ M, the concentration of N-acetyl-L-cysteine is 1 mM, the concentration of porcine intestinal mucosal heparin sodium is 10 μ g/mL, the concentration of water-soluble vitamin E is 10 μ M, AXL inhibitor R428 is 2 μ M, the concentration of hepatocyte growth factor is 50 ng/mL, the concentration of 5 hydroxytryptamine is 10 μ M, the concentration of beta-cell is 20 zxft 5334/L, the concentration of laryngeal peptide is ng/L, and the concentration of serine is 10 μ M4264/mL.

The seventh stage induced differentiation medium of 9-11 days in the group B of the present stage consists of 50% Ham's F-12 medium, 50% medium 199, glutamine, calcium chloride dihydrate, N-2 hydroxyethyl piperazine-N-2-ethanesulfonic acid, defatted BSA (bovine serum albumin) (BSA), insulin-transferrin-selenium, ethanolamine, water-soluble vitamin E, nicotinamide, heparin sodium, deoxyribonuclease I, necrotizing apoptosis inhibitor Necrostatin-1, and serine protease inhibitor Pefabloc, and the solution is changed half a day.

In the culture medium, glutamine concentration of 2 mM, two water calcium chloride concentration of 2.5 mM, N-2 hydroxyethyl piperazine-N-2-ethane sulfonic acid concentration of 10 mM, defatted BSA (fetal Bovine Serum Albumin) (BSA) concentration of 2%, insulin-transferrin-selenium concentration of 0.6 mL/L, ethanolamine concentration of 1 mg/L, water soluble vitamin E concentration of 10 μ M, nicotinamide concentration of 10 mM, heparin sodium concentration of 10 μ g/mL, deoxyribonuclease I concentration of 1U/mL, necrotizing apoptosis inhibitor Necrostatin-1 concentration of 100 μ M, serine protease inhibitor Pefabloc concentration of 0.1 μ M.

Example 2 qPCR detection of mRNA expression level of marker gene at each induced differentiation stage and immunofluorescence detection of cell positivity at each induced differentiation stage

1. Experimental methods

1.1 RNA extraction

(1) Supplementing 500 mu L Trizol solution to the collected islet cell sample, cracking for 5 min at room temperature, adding 200 mu L chloroform, violently shaking and uniformly mixing 15 s, and incubating for 3 min at room temperature;

(2) After centrifugation at 12000 rpm for 10 minutes at 4 ℃ the sample will separate into three layers: the lower organic phase, the middle layer and the upper colorless aqueous phase, RNA is present in the aqueous phase. The capacity of the aqueous layer was about 60% of the volume of RL added, the aqueous phase was transferred to a new tube and the next operation was carried out;

(3) 1-fold volume of 70% ethanol was added (please first check if absolute ethanol had been added!), and the mixture was mixed by inversion (precipitation may occur). Transferring the obtained solution and possible precipitate into adsorption column RA, centrifuging at 10,000 rpm for 45 s, discarding waste liquid, and sleeving the adsorption column back to the collecting tube;

(4) Adding 500 mu L of deproteinized liquid RE, centrifuging at 12,000 rpm for 45 seconds, and discarding waste liquid;

(5) Adding 700 μ L of rinsing solution RW (please check whether absolute ethanol is added), centrifuging at 12,000 rpm for 60 s, and discarding the waste solution;

(6) Adding 500 μ L of rinsing solution RW, centrifuging at 12,000 rpm for 60 s, and discarding the waste liquid;

(7) Putting the adsorption column RA back into an empty collection pipe, centrifuging at 12,000 rpm for 2 minutes, and removing the rinsing liquid as much as possible so as to prevent residual ethanol in the rinsing liquid from inhibiting downstream reaction;

(8) Taking out the adsorption column RA, placing into an RNase free centrifuge tube, and adding 50 μ L of RNase free wall (incubated at 65-70 deg.C in advance) at the middle part of the adsorption membrane according to the expected RNA yield;

(9) The mixture was left at room temperature for 2 minutes and centrifuged at 12,000 rpm for 1 minute. If more RNA is needed, the obtained solution can be added into the centrifugal adsorption column again and centrifuged for 1 minute, or 30 μ L of RNase free water is added additionally and centrifuged for 1 minute, and the two eluates are combined;

(10) The larger the elution volume, the higher the elution efficiency, and if the RNA concentration is required to be high, the elution volume can be reduced appropriately, but the minimum volume is preferably not less than 50. Mu.L, and the smaller the volume, the lower the RNA elution efficiency, and the RNA yield.

1.2 Reverse transcription

(1) Calculating according to the total RNA concentration, and reversing according to the concentration of 1 mu g;

(2) Reagents were premixed with total RNA according to the reaction system described in table 3 below;

(3) Transferring the pre-mixed solution to a PCR instrument, and carrying out reaction by using a cDNA template (incubation at 42 ℃ for 30 min, and incubation at 85 ℃ for 5 s);

(4) Rapidly transferring the reversed cDNA onto ice for 1 min for cooling;

(5) Storing at-20 deg.C, and diluting according to required concentration before use. (Note: check if the centrifuge tube is covered before putting into the PCR apparatus to prevent high temperature evaporation; check if the centrifuge tube is broken by high temperature after taking out from the PCR apparatus.

1.3 qPCR experimental method

1.3.1 Primer testing

(1) The specific reaction system and the reaction conditions such as formal experiments need to be tested by qPCR before the formal experiments of the primers designed according to DNA, and each pair of primers needs to be subjected to template water control.

(2) After obtaining the result, firstly, judging the specificity of the primer according to the melting curve, wherein the selection standard is as follows: single peak and narrow peak shape, no obvious primer dimer melting curve peak in water control. If the designed melting curves of the multiple pairs of primers show good specificity, primers with smaller Ct value and higher amplification efficiency are preferentially selected to be applied to the amplification curve of each primer for formal experiments. Specific information of primer sequences for qPCR detection of genes is shown in Table 4.

1.3.2 Sample discharge

(1) The sample application of one sample is arranged in the same row as much as possible;

(2) If three replicates in a formal experiment cannot be arranged on the same plate, the experiments in the three replicates need to be separated, but the experiments in the same replicate cannot be separated into two plates.

1.3.3 Formal experiment

(1) Premixing was performed according to the reaction system shown in the following table 5;

(2) Adding the premixed reagent into an eight-connected tube, wherein each hole is 18 mu L;

(3) Quickly adding 2 mu L of cDNA into the eight-connected tubes, covering the eight-connected tubes with a cover, and placing each row of eight-connected tubes on a palm centrifuge for centrifugation for several seconds;

(4) Putting the mixture into a Light cycler instrument to react according to a 3-step method, wherein the cycle number is 40;

the templates are shown in table 6 below;

note that: the cDNA is diluted with sterilized pure water 1:4, and if a sample with low gene expression is encountered, the cDNA is arranged in a certain sequence and then added into a just prepared reaction system. After the sample is added, the eight-tube-connected cover is covered, and the 1-12 sequence is marked on the edge of the uppermost edge of the eight-tube-connected cover (the mark cannot be written on the cover of the reaction tube, the eight-tube-connected cover prevents a naked hand from touching the transparent fluorescence collection area in the middle, and ensures that each hole is covered tightly, otherwise, the repeatability is influenced or the peak drift of the melting curve can occur.

1.3.4 Upper machine

Turning on a power switch of the qPCR instrument; opening the sample frame, putting the eight connecting pipes, closing the sample frame, and setting a template; clicking a Start key to Start running a program; after the program is run, taking out the eight connecting pipes on the sample rack; marking the sample name and the name of the detection gene of each reaction hole on software, and storing result files in a classified manner; after the reaction is finished, the eight connecting tubes are filled into a sealing bag for storage.

1.4 Immunofluorescence

(1) Cleaning cells at each stage for identification with DPBS for three times, fixing 4% PFA at room temperature for 40 min, and keeping out of the sun (recovering PFA at room temperature for reuse);

(2) DPBS washing for three times, 0.5% TritonX-100 (DPBS dissolving), and perforating for 20 min;

(3) 5% BSA blocking solution +0.1% TritonX-100,4 deg.C overnight blocking;

preparing a solution: PBST-1 (for washing): DPBS +0.1% TritonX-100; PBST-2 (for primary antibody and secondary antibody): DPBS +0.1% TritonX-100+1% BSA;

incubating primary antibody, wherein PBST-2=1, and the temperature is kept overnight at 4 ℃;

recovering the primary antibody solution, and washing with PBST-1 shaking table for 10 min for three times;

the incubation secondary antibody, PBST-2=1, overnight at 4 ℃ and protected from light. (the driving time can also be 1 h at room temperature, shaking table) from the adding of the fluorescent secondary antibody, all the following operation steps are processed in a dark place;

PBST-1 is cleaned for 10 min for three times by a shaking table;

DAPI dilution 1000 Xincubation for 2-3 min, light protection. Cleaning the DPBS 1~2 times;

and directly photographing and observing.

2. Results of the experiment

(1) The first stage test results are shown in fig. 2A and 2B. The results show that the induced differentiation of iPSC cells changes morphology, the cell density continues to increase at this stage, and cell death is also accompanied, when the nucleoplasmic ratio of endoderm cells is significantly reduced compared to undifferentiated stem cells, and there is a clear boundary where the cells are in contact;

after the iPSC is induced, SOX17 and FOXA2 are used as markers of endoderm, the positive rate of the endoderm is higher than 50%, in the embodiment of the invention, a GDF-8 group is used as a B group, activin A is used as an A group, and qPCR detection shows that SOX17 gene expressed by the A group is 4.5 times of that of the B group and 500 times of that of a negative control group iPSC; the FOXA2 gene expressed by the group A is 7 times of that of the group B and 110 times of that of the negative control group iPSC; the CRCX4 gene expressed by the group A is 2 times of that of the group B and is 3 times of that of the negative control group iPSC. Expression was >50% by detection of FOXA2/DAPI (immunofluorescence). The induction efficiency is higher when the iPSC is induced by combining Activin A and CHIR-99021, the number of the obtained endoderm cells is larger, the positive rates of SOX17 and FOXA2 are higher, and the induction effect is obviously higher than that of GDF-8.

(2) The second stage test results are shown in fig. 3A and 3B. The results showed that the induced differentiation of iPSC cells changed morphology, and cord-like, globular cells began to appear at the end of this phase. The expression of cord-shaped and spherical cell mass positive cells is highest;

at this stage, HNF1 beta and FOXA2 are used as markers of the primitive gut tube, the positive rate of the primitive gut tube is higher than 60%, and qPCR detection shows that the positive rate of HNF1 beta gene is about 60% and the positive rate of FOXA2 gene is about 80% of endoderm cells obtained after induction.

(3) The detection results of the third stage are shown in fig. 4A and fig. 4B, and the results show that the morphology of the induced and differentiated iPSC cells is changed, and the cord-shaped and spherical cell masses are continuously increased at the stage;

PDX-1 and SOX9 are used as markers of the posterior foregut stage at the stage, the positive rate of the posterior foregut stage is higher than 70%, qPCR detection shows that the positive rate of PDX-1 gene is higher than 60% and the positive rate of SOX9 gene is higher than 25%.

(4) The detection results of the fourth stage are shown in fig. 5A and fig. 5B, and the results show that the cell morphology after induced differentiation is changed, and the spherical structure continues to be formed in a large amount and becomes compact gradually at this stage;

PDX-1 and SOX9 are used as markers of the posterior foregut stage at the stage, the positive rate of the posterior foregut stage is higher than 70%, qPCR detection shows that the positive rate of PDX-1 gene is higher than 90% and the positive rate of SOX9 gene is higher than 50%.

(5) The fifth stage test results are shown in fig. 6. The result shows that the iPSC cell after induced differentiation changes in morphology, the cells continue to grow in a stacked manner, and the spherical structure is connected into a sheet;

in this stage, NKX6.1, NGN3 and PDX-1 were used as the endocrine progenitor phenotype, and it was found that the expression level of NKX6.1 mRNA, the expression level of NGN3 mRNA and the expression level of PDX-1 mRNA were about 50 times, 80 times and 200 times, respectively, of the marker gene of the cells induced in the fifth stage.

(6) The results of the sixth and seventh stage tests are shown in FIGS. 7A and 7B, and show that INS, GCG, NKX6.1, and MAFA are used as islet cell phenotype in this stage, the expression level of NKX6.1 mRNA, which is a marker gene in group B, is about 1.1 times that in group A, the expression level of GCG mRNA, which is a marker gene in group B, is about 2 times that in group A, and the expression level of INS mRNA, which is a marker gene in group B, is about 17 times that in group A. Immunofluorescence results show that the single positive rate of INS in group B is about 95%, and the single positive rate of INS in group A and GCG are co-expressed, namely the single positive rate of GCG-/INS + cells in group B is obviously improved.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

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Claims

1. An induction differentiation agent for inducing iPSCs to differentiate into functionally mature islet beta cells, which is characterized by comprising a first-stage induction differentiation agent, a second-stage induction differentiation agent, a third-stage induction differentiation agent, a fourth-stage induction differentiation agent, a fifth-stage induction differentiation agent, a sixth-stage induction differentiation agent and a seventh-stage induction differentiation agent;

the first-stage induced differentiation agent comprises a first-stage induced differentiation agent A and a first-stage induced differentiation agent B;

the first stage induction differentiation agent A comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, activin A and CHIR-99021;

the first-stage induction differentiation agent B comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin and Activin A;

the second stage induction differentiation agent comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ascorbic acid and FGF-7;

the third stage induction differentiation agent comprises sodium bicarbonate, glutamine, glucose, bovine serum albumin, ascorbic acid, FGF-7, insulin-transferrin-selenium, ethanolamine, SANT-1, retinol, LDN193189, TPPB;

the fourth stage induction differentiation agent comprises sodium bicarbonate, glutamine, glucose, bovine serum albumin, ascorbic acid, FGF-7, insulin-transferrin-selenium, ethanolamine, SANT-1, retinol, LDN193189, TPPB;

the fifth stage induction differentiation agent comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, SANT-1, retinol, LDN193189, triiodothyronine (T3), ALK5i II, and zinc sulfate;

the sixth stage induction differentiation agent comprises a sixth stage induction differentiation agent A, a sixth stage induction differentiation agent B, a sixth stage induction differentiation agent C and a sixth stage induction differentiation agent D;

the sixth stage induction differentiation agent A comprises beta-cytomin, latrunculin A, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, LDN193189, triiodothyronine (T3), ALK5i II, zinc sulfate, GSi XX;

the sixth stage induction differentiation agent B comprises beta-cytomin, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, LDN193189, triiodothyronine (T3), ALK5i II, zinc sulfate, GSi XX;

the sixth stage induction differentiation agent C comprises betacellulin, forskolin, exenatide 4, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, LDN193189, triiodothyronine (T3), ALK5i II, zinc sulfate, GSi XX;

the sixth stage induction differentiation agent D comprises betacellulin, forskolin, exenatide 4, hepatocyte growth factor, 5 hydroxytryptamine, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ethanolamine, LDN193189, triiodothyronine (T3), ALK5i II, zinc sulfate, GSi XX, insulin-transferrin-selenium;

the seventh stage induction differentiation agent comprises a seventh stage induction differentiation agent A and a seventh stage induction differentiation agent B;

the seventh stage induction differentiation agent A comprises beta-cytomin, forskolin, exenatide 4, hepatocyte growth factor, 5 hydroxytryptamine, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, triiodothyronine (T3), ALK5i II, zinc sulfate, N-acetyl-L-cysteine, porcine intestinal mucosa heparin sodium, water-soluble vitamin E, R;

the seventh stage induction differentiation agent B comprises glutamine, calcium chloride dihydrate, N-2 hydroxyethyl piperazine-N-2-ethane sulfonic acid, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, water-soluble vitamin E, nicotinamide, heparin sodium, deoxyribonuclease I, necrostatin-1 and Pefabloc;

the concentrations of all components in the first-stage induction differentiation agent are respectively as follows: 100 ng/mL Activin A, 3. Mu.M CHIR-99021, 0.5% bovine serum albumin, 1.5g/L sodium bicarbonate, 10 mM glucose, 1 mM glutamine;

the concentrations of all components in the second stage induction differentiation agent are respectively as follows: 0.25 mM ascorbic acid, 50 ng/mL FGF-7, 0.5% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, 10 mM glucose, 1 mM glutamine;

the concentration of each component in the third stage induction differentiation agent is as follows: 0.25 mM ascorbic acid, 50 ng/mL FGF-7, 0.25 μ M SANT-1, 1 μ M retinol, 100 nM LDN193189, 200 nM TPPB, 2% fetal bovine serum albumin, 2.5 g/L sodium bicarbonate, 10 mM glucose, 1 mM glutamine, 1 mg/L ethanolamine, 0.5% insulin-transferrin-selenium;

the concentration of each component in the fourth-stage induction differentiation agent is respectively as follows: 0.25 mM ascorbic acid, 2 ng/mL FGF-7, 0.25 μ M SANT-1, 0.1 μ M retinol, 200 nM LDN193189, 100 nM TPPB, 2% fetal bovine serum albumin, 2.5 g/L sodium bicarbonate, 10 mM glucose, 1 mM glutamine, 1 mg/L ethanolamine, 0.5% insulin-transferrin-selenium;

the concentrations of all components in the inducing and differentiating agent in the fifth stage are respectively as follows: 0.25 μ M SANT-1, 0.05 μ M retinol, 100 nM LDN193189, 1 μ M triiodothyronine (T3), 10 μ M ALK5i II,10 μ M zinc sulfate, 2% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, 20 mM glucose, 1 mM glutamine, 0.5% insulin-transferrin-selenium, 1 mg/L ethanolamine;

the concentrations of all components in the inducing and differentiating agent in the sixth stage are respectively as follows: 100 nM LDN193189, 1. Mu.M triiodothyronine (T3), 10. Mu.M ALK5i II, 10. Mu.M zinc sulfate, 2% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, 20 mM glucose, 1 mM glutamine, 1 mg/L ethanolamine, 100 nM GSi XX, 1. Mu.M latrunculin A, 50 ng/mL hepatocyte growth factor, 10. Mu.M 5 hydroxytryptamine, 20 ng/mL betacellulin, 10. Mu.M forskolin, 50 ng/mL exenatide 4;

the concentration of each component in the induction differentiation agent A in the seventh stage is respectively as follows: 2% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, 20 mM glucose, 1 mM glutamine, 1 mg/L ethanolamine, 0.5% insulin-transferrin-selenium, 1 μ M triiodothyronine (T3), 10 μ M ALK5i II,10 μ M zinc sulfate, 1 mM N-acetyl-L-cysteine, 10 μ g/mL porcine mucosal heparin sodium, 10 μ M water-soluble vitamin E, 2 μ M R428, 50 ng/mL hepatocyte growth factor, 10 μ M5 hydroxytryptamine, 20 ng/mL beta cell, 10 μ M forskolin, 50 ng/mL exenatide 4;

the concentration of each component in the induction differentiation agent B in the seventh stage is respectively as follows: 2 mM glutamine, 2.5 mM calcium chloride dihydrate, 10 mM N-2 hydroxyethyl piperazine-N-2 ethane sulfonic acid, 2% fetal bovine serum albumin, 0.6 mL/L insulin-transferrin-selenium, 1 mg/L ethanolamine, 10 μ M water soluble vitamin E, 10 mM nicotinamide, 10 μ g/mL heparin sodium, 1U/mL deoxyribonuclease I, 100 μ M Neostatin-1, 0.1 μ M Pefabloc.

2. An induced differentiation medium for inducing iPSCs to differentiate into functionally mature islet beta cells is characterized by comprising a first-stage induced differentiation medium, a second-stage induced differentiation medium, a third-stage induced differentiation medium, a fourth-stage induced differentiation medium, a fifth-stage induced differentiation medium, a sixth-stage induced differentiation medium and a seventh-stage induced differentiation medium;

the first stage induced differentiation medium comprises a basal medium MCDB131, the first stage induced differentiation agent as described in claim 1;

the second-stage induced differentiation medium comprises a basal medium MCDB131, a second-stage induced differentiation agent as described in claim 1;

the third-stage induced differentiation medium comprises a basal medium MCDB131 and a third-stage induced differentiation agent as described in claim 1;

the fourth stage induced differentiation medium comprises a basal medium MCDB131, a fourth stage induced differentiation agent as described in claim 1;

the fifth stage induced differentiation medium comprises a basal medium MCDB131, a fifth stage induced differentiation agent as described in claim 1;

the sixth stage induced differentiation medium comprises a basal medium MCDB131 and the sixth stage induced differentiation agent as described in claim 1;

the seventh stage induced differentiation medium comprises a basal medium MCDB131, 50% Ham's F-12 medium, 50% medium 199, the seventh stage induced differentiation agent described in claim 1.

3. A method of inducing the differentiation of iPSCs into functional mature pancreatic islet beta cells, comprising the steps of:

(2) A first stage of induced differentiation, which is to use the first stage induced differentiation culture medium of claim 2 to induce the iPSCs obtained in the step (1) to differentiate towards definitive endoderm cells;

(3) Inducing differentiation of definitive endoderm cells to primitive gut tube cells by using the second-stage induction differentiation medium according to claim 2;

(4) A third stage of inducing differentiation, wherein the third stage of inducing differentiation culture medium described in claim 2 is used for inducing differentiation of the gastrula cells to the retroforegut cells;

(5) A fourth step of inducing differentiation, wherein the fourth step of inducing differentiation medium according to claim 2 is used to induce differentiation of the pre-intestinal cells into pancreatic progenitor cells;

(6) Inducing differentiation at a fifth stage, inducing differentiation of pancreatic progenitor cells into pancreatic endocrine progenitor cells using the fifth stage induction differentiation medium according to claim 2;

(7) A sixth stage of inducing differentiation, wherein the differentiation of pancreatic endocrine progenitor cells into pancreatic endocrine cells is induced by using the sixth stage of inducing differentiation medium according to claim 2;

(8) A seventh stage of induced differentiation, wherein the seventh stage of induced differentiation medium according to claim 2 is used for inducing pancreatic endocrine cells to differentiate into mature islet cells, so as to obtain functional mature islet beta cells;

the culture time in the step (1) is 3 days;

day-3 to Day-1, replacing the fresh medium every Day;

the time for inducing differentiation in the first stage is 3 days;

on the induction day1, adopting a first-stage induction differentiation culture medium A to perform induction differentiation;

on the induction days 2-3, adopting a first-stage induction differentiation culture medium B to perform induction differentiation;

day0-Day2, replacing fresh culture medium every Day;

the time for inducing differentiation in the second stage is 2 days;

on induction days 1-2, adopting the second-stage induced differentiation culture medium to perform induced differentiation;

day3-Day4, change fresh medium every Day;

the time for inducing differentiation in the third stage is 2 days;

on the induction 1-2 days, adopting the third stage induction differentiation culture medium to carry out induction differentiation;

day5-Day6, change fresh medium every Day;

the fourth stage induction differentiation time is 3 days;

on induction days 1-3, adopting the fourth stage induction differentiation culture medium to carry out induction differentiation;

day7-Day9, change fresh medium every Day;

the time for inducing differentiation in the fifth stage is 3 days;

on induction days 1-3, adopting the fifth stage induction differentiation culture medium to carry out induction differentiation;

day10-Day12, change fresh medium every Day;

the time for inducing differentiation in the sixth stage is 14 days;

on the induction day1, adopting a sixth stage induction differentiation culture medium A to carry out induction differentiation;

on the induction days 2-7, adopting a sixth stage induction differentiation culture medium B to carry out induction differentiation;

on the 8 th to 9 th days of induction, adopting a sixth stage induction differentiation culture medium C for induction differentiation;

on induction days 10-14, inducing differentiation with sixth stage inducing differentiation culture medium D;

day13-Day27, half a Day for liquid change;

the time for inducing differentiation in the seventh stage is 14 days;

on induction days 1-8, adopting the seventh stage induction differentiation culture medium A to carry out induction differentiation;

on induction days 9-11, adopting the seventh stage induction differentiation culture medium B to carry out induction differentiation;

day28-Day40, half a Day.

4. The method according to claim 3, wherein the complete medium in step (1) is E8 complete medium;

the E8 complete medium contains a ROCK inhibitor;

the ROCK inhibitor is Y27632;

the concentration of Y27632 was 10. Mu.M.

5. The method according to claim 4, wherein the iPSCs of step (1) are derived from humans.

6. Use of the induction differentiation agent according to claim 1 for preparing an induction differentiation medium for inducing differentiation of iPSCs into functional islet beta cells.

7. The use of the induction differentiation agent as defined in claim 1 for inducing the differentiation of iPSCs into functional islet beta cells.

8. The use of the differentiation-inducing medium according to claim 2 for inducing differentiation of iPSCs into functional islet beta cells.